Regulation of ethanol-related behavior and ethanol metabolism by the Corazonin neurons and Corazonin receptor in Drosophila melanogaster

Abstract

Impaired ethanol metabolism can lead to various alcohol-related health problems. Key enzymes in ethanol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH); however, neuroendocrine pathways that regulate the activities of these enzymes are largely unexplored. Here we identified a neuroendocrine system involving Corazonin (Crz) neuropeptide and its receptor (CrzR) as important physiological regulators of ethanol metabolism in Drosophila. Crz-cell deficient (Crz-CD) flies displayed significantly delayed recovery from ethanol-induced sedation that we refer to as hangover-like phenotype. Newly generated mutant lacking Crz Receptor (CrzR(01) ) and CrzR-knockdown flies showed even more severe hangover-like phenotype, which is causally associated with fast accumulation of acetaldehyde in the CrzR(01) mutant following ethanol exposure. Higher levels of acetaldehyde are likely due to 30% reduced ALDH activity in the mutants. Moreover, increased ADH activity was found in the CrzR(01) mutant, but not in the Crz-CD flies. Quantitative RT-PCR revealed transcriptional upregulation of Adh gene in the CrzR(01) . Transgenic inhibition of cyclic AMP-dependent protein kinase (PKA) also results in significantly increased ADH activity and Adh mRNA levels, indicating PKA-dependent transcriptional regulation of Adh by CrzR. Furthermore, inhibition of PKA or cAMP response element binding protein (CREB) in CrzR cells leads to comparable hangover-like phenotype to the CrzR(01) mutant. These findings suggest that CrzR-associated signaling pathway is critical for ethanol detoxification via Crz-dependent regulation of ALDH activity and Crz-independent transcriptional regulation of ADH. Our study provides new insights into the neuroendocrine-associated ethanol-related behavior and metabolism.

Figure 1. Hangover-like phenotype of Crz-CD.

Crz neuron ablation induced by hid (A) and rpr (B) transgene expression (triangles) leads to delayed and incomplete recovery compared to wild-type (circles). Each data point is a mean ± sem (n = 3–5). All genotypes are in y w background. (C) Flies recovered from ethanol-induced sedation at 2 hours after exposure. (1, Crz-gal4/+; 2, UAS-hid/+; 3, Crz-gal4/UAS-hid).

Figure 2. Generation of CrzR-null allele.

(A) Diagram of CrzR encoding exons and approximate locations of PCR primers. Exons and introns are shown as arrows and solid lines, respectively. Coding exons are shown in grey, and UTRs in white. An 8-kb deletion was indicated by the hatched box. A footprint of gal4 coding region and flanking sequence derived from the Minos element was found at the original insertion site. (B) PCR products derived from the designated primer sets and mutant genomic DNA. No specific PCR product was produced from f3 primer. (C) RT-PCR. CrzR⁰¹ did not produce PCR product, while the wild-type did it with expected size.

Figure 3. Hangover-like phynotype of the CrzR⁰¹ mutant flies.

Flies were exposed to 100% ethanol as indicated. (A) Homozygous CrzR⁰¹. (genotypes: y w;; CrzR⁰¹) (B) CrzR⁰¹/Df. (genotypes: w¹¹¹⁸;; +/Df: w¹¹¹⁸;; CrzR⁰¹/Df). (C) Same as in (B), except for 18-min exposure period. Each data point represents mean ± sem (n = 5).

Figure 4. Lack of Crz/CrzR leads to reduced ALDH activity.

(A) Levels of acetaldehyde in adult males exposed to 100% ethanol. (n = 4). (B) CrzR⁰¹ mutation resulted in the reduction of whole cell ALDH activity (n = 3). (C) Succinate dehydrogenase activity in mitochondria (Mt) and cytosol (Ct). The activity was detected almost exclusively in the mitochondrial fraction (n = 3). (D) Reduction of mitochondrial ALDH activity (AL) in CrzR⁰¹ mutant. Succinate dehydrogenase activity (SD) is similar between control and mutant. (n = 3). (E, F) Crz-CD leads to reduced ALDH activity. (E) Whole cell ALDH activities of Crz::hid are significantly lower than those of the transgenic controls (n = 8). (F) grim-induced Crz-CD produced significant reduction of mitochondrial ALDH activity (n = 3). (*P<0.05; **P<0.01; ***P<0.001; ns, not significant). Each data point represents mean ± sem for the indicated replicates. All genotypes are in y w background.

Figure 5. CrzR mutation causes significant increase of ADH activity.

(A) Survival rates of CrzR⁰¹ (dashed lines) and wild-type (solid line) in response to acetaldehyde as indicated (n = 3). (B) Histogram showing ADH activity (n = 4). (C,D) ADH activity in Crz-CD. (C) hid- and (D) grim-induced Crz cell ablation did not increase the whole cell ADH activity (P > 0.05, n  =  3). (*** P<0.001). Each data point represents mean ± sem for the indicated replicates. All genotypes are in y w background.

Figure 6. CrzR regulates Adh mRNA levels through PKA-dependent pathway.

(A) RT-qPCR using two different sets of primers revealed 2.7-fold increase of Adh transcript level in CrzR⁰¹ compared to the wild-type (n = 4). (B) No significant change of Aldh transcript level in CrzR⁰¹ (n = 4). (C,D) Expression of PKA inhibitor (C) or dominant negative CREB (D) using CrzR-gal4 induced severe hangover-like phenotype (n = 5). (E) PKA inhibition in CrzR cells increased ADH activity by 50% compared to the controls (n = 3). (F) PKA inhibition in CrzR cells leads to significant increase of Adh transcript level (n = 3). (G) No delayed recovery was observed from flies expressing PKC inhibitor from CrzR-Gal4 (P > 0.05, n  =  5). (H) Inhibition of PKC did not affect ADH activity (p > 0.05, n  =  3). (**P<0.005; *** P<0.001). Each data point represents mean ± sem for the indicated replicates. All genotypes are in y w background.

Figure 7. CrzR-gal4 driven reporter gene expression.

UAS-mCD8GFP;; UAS-Redstinger was expressed under the control of three CrzR-gal4 drivers as indicated. (A-C) Third instar larva. (A) CNS. (B) salivary gland. (C) fat body. (D) Adult fat tissue. Scale bar = 50 µm. (green, membrane bound GFP; red, nuclear RFP).